US4396606A - Novel polypeptide analgesics - Google Patents

Novel polypeptide analgesics Download PDF

Info

Publication number
US4396606A
US4396606A US06/091,615 US9161579A US4396606A US 4396606 A US4396606 A US 4396606A US 9161579 A US9161579 A US 9161579A US 4396606 A US4396606 A US 4396606A
Authority
US
United States
Prior art keywords
arg
leu
amino acids
lys
gly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
US06/091,615
Other languages
English (en)
Inventor
Avram Goldstein
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Research Corp Technologies Inc
Original Assignee
Addiction Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Addiction Research Foundation filed Critical Addiction Research Foundation
Priority to US06/091,615 priority Critical patent/US4396606A/en
Priority to DE8080303698T priority patent/DE3066200D1/de
Priority to EP80303698A priority patent/EP0029300B1/en
Priority to CA000363326A priority patent/CA1177825A/en
Priority to JP15394380A priority patent/JPS5681550A/ja
Application granted granted Critical
Publication of US4396606A publication Critical patent/US4396606A/en
Assigned to RESEARCH CORPORATION reassignment RESEARCH CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: ADDICTION RESEARCH FOUNDATION
Assigned to RESEARCH CORPORATION TECHNOLOGIES, INC. reassignment RESEARCH CORPORATION TECHNOLOGIES, INC. ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: RESEARCH CORPORATION
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9486—Analgesics, e.g. opiates, aspirine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the first five amino acids of ⁇ -endorphin are the five amino acids of met-enkephalin.
  • analgesic In developing new polypeptide analgesics, there are a number of important considerations. A desirable analgesic will have relatively few amino acids, so as to be easily and economically synthesized in good yield. Furthermore, it is desirable that the polypeptide analgesic be easily administered and migrate to its natural binding site without substantial degradation. Otherwise, the polypeptide will have to be introduced adjacent to or at the site of binding to achieve the analgesic effect. Desirably, the potency should be substantially higher than available analgesics, while at the same time because of the reduced amount administered or because of its particular structure, the analgesic should be relatively free of undesirable side effects.
  • Novel oligopeptides having alternating basic hydrophilic amino acids and hydrophobic amino acids, having at least five units, which oligopeptides are employed as precursors for conjugating to opioid compounds, particularly polypeptide opioids.
  • the oligopeptides are joined by a short chain to a phenolic group, which may be part of a tyrosyl unit or a morphine alkaloid or synthetic mimetic analogs thereof.
  • polypeptude having at the N-terminus a tridecapeptide having at the N-terminus the five amino acid sequence of leu-enkephalin and an octapeptide which includes the alternating sequence.
  • the subject compounds find use as precursors to analgesics, as analgesics, as opioid agonists and in studying the structure of opioid binding sites.
  • the subject polypeptides may be combined with acidic polypeptides having reciprocal amino acids to provide substantially neutral salts.
  • compounds are provided for use in binding to opioid receptor sites of mammals which involve an oligopeptide pentet, usually sextet, of alternating basic hydrophilic side chains and hydrophobic side chains or hydrogen.
  • the oligopeptide is desirably linked to a phenolic group through a relatively short chain, either an amino acid chain generally having at least one amino acid, usually having from about 3 to 6 amino acids, or by at least about 3 atoms and not more than 18 atoms, the atoms in the chain being C and N, while the linking group comprising the chain and atoms bonded thereto consisting of C, H, N and O, where O is oxy or non-oxo-carbonyl, N is amino or amido and C is aliphatic (includes cycloaliphatic).
  • the oligopeptide may be bonded to compounds having known opioid activity, such as morphine, leu-enkephalin, or met-enkephalin, at a site distant from the phenolic group.
  • the opioid compounds will have molecular weights of at least about 1,200, usually 1,500 and not more than about 2,500. They will generally have from about 4 to 6, usually 5, basic amino acid groups, not more than a total of four basic amino acids linked to another basic amino acid.
  • the oligopeptide will have the following formula. ##STR1## wherein: one of a and b is 0 or 1 and the other is 1;
  • all the Rs and Ds are bonds or alkylene groups of from 1 to 6, preferably from 1 to 4, more preferably from 2 to 3 carbon atoms wherein the alkylene groups may be straight or branched chain, usually methylene, polymethylene or ( ⁇ -1)methylpolymethylene with the proviso that the Rs and Ds are alkylene when bonded to other than hydrogen;
  • either the Xs or the Ys are hydrogen, when other than hydrogen, they are basic nitrogen groups, normally amino, amidinyl or guanidinyl;
  • a 1 may be taken together with X b or when the Ys are hydrogen, A 2 may be taken together with Y b and the atoms to which they are attached to form a heterocyclic ring of from five to six members, which may be hydroxyl substituted or unsubstituted, normally unsubstituted;
  • a 1 X b or A 2 Y b defining an alkylene or hydroxylkylene chain; when not taken together with X b or Y b , A 1 and A 2 are hydrogen; and W is OH, amino or an amino acid or polypeptide of up to about ten, usually up to about four, amino acids, which do not interfere with the opioid properties of the base structure and are joined by a peptide bond.
  • R a -X a When the Xs are hydrogen, R a -X a will be alkyl of from 1 to 5, preferably of from 2 to 3 carbon atoms, more preferably isopropyl;
  • R b -X b is alkylene of from 1 to 5, usually of from 2 to 3 carbon atoms and particularly preferred that R b -X b is taken together with A 1 to provide trimethylene or hydroxytrimethylene;
  • R c -X c is alkylene of from 1 to 5, more usually of from 2 to 4 carbon atoms, and preferably propyl;
  • D a -Y a is a terminal basic substituted alkylene group of from 1 to 5, preferably 2 to 3 carbon atoms, where Y a is amino or guanidinyl, preferably D a is alkylene of three carbon atoms and Y a is guanidinyl;
  • D b -Y b is a terminal basic substituted alkylene group of from 1 to 5, more usually of from 2 to 4 carbon atoms, and preferably 4 carbon atoms, and Y b is amino;
  • D c -Y c has the same definition as D b -Y b .
  • R c -X c has the definition of D c -Y c previously indicated.
  • a preferred embodiment of Formula 1 is a polypeptide of at least six, preferably seven amino acids, where the polypeptide embodies the amino acids of Formula 1, and has two additional amino acids at the N-terminus, preferably at least one, and more preferably both being basic amino acids and the N-terminus of the Formula 1 oligopeptide is hydrophobic, i.e. X a is hydrogen.
  • D d-e are a bond (only when the Y to which the D is bonded is hydrogen) or alkylene groups of from 1 to 6, usually from 2 to 5, preferably from 2 to 4 carbon atoms and Y d-e are hydrogen or a basic nitrogen group e.g., amino, amidinyl or guanidinyl, particularly, D d-e are alkylene of three carbon atoms and at least one of Y d-e is amino or guanidinyl; preferably all the Ys are basic groups;
  • W 1 is hydroxy, amino or an amino acid or polypeptide of up to about 10, usually 4, amino acids bonded by a peptide link to Formula 1;
  • p is 0 or 1, usually 1, and a and b are preferably 1.
  • Both Formula 1 and Formula 2 may be modified in a number of different ways.
  • One or more, usually not more than 2, and preferably only 1 may be other than the natural L-configuration.
  • the unnatural D-configuration will be a hydrophobic amino acid, rather than a basic hydrophilic amino acid.
  • the amino groups may be modified by acetylation or nitro-substituted to reduce the basicity of the molecule, or may be employed as mineral acid salts, such as the hydrochlorides.
  • Either Formula 1 or Formula 2 may be bonded to a compound having opioid activity.
  • opioid activity is intended agonist, antagonist and partial agonist activity.
  • Opioid activity is recognized by effectiveness in such common tests as the guinea pig ileum and the vas deferens tests described subsequently.
  • An antagonist is a compound which is not effective in such tests, but is able to block a compound which is effective.
  • a partial agonist is a compound which shows both effects, having some effectiveness in the test, but able to block other effective compounds. In effect, all of these compounds are ligands for an opioid receptor.
  • Various compounds having opioid activity may be employed, particularly those having a phenolic group or a substituted phenolic group, where the substituent may be removed in vivo.
  • Such compounds include morphine, heroin, codeine, naloxone, levorphanol, nalorphine, naltrexone, leu-enkephalin and met-enkephalin.
  • the morphine compounds will for the most part have the following formula: ##STR3## wherein: one of Z or Z 1 is a linking group to Formula 1 or Formula 2 of from 1 to 10, usually 1 to 6 carbon atoms; when not a linking group, Z is hydroxyl and Z 1 is an aliphatic group (includes cycloaliphatic) of from 1 to 4, usually 1 to 3, carbon atoms having from 0 to 1 site of ethylenic unsaturation, normally methyl, allyl or cyclopropylmethyl;
  • b is hydrogen or methyl
  • Q is hydrogen or hydroxyl
  • W 2 is hydroxyl, amino or an amino acid or polypeptide of not more than about 10, usually 4, amino acids which does not adversely affect the opioid activity of the subject oligopeptides.
  • compositions are those related to enkephalin derivatives, to the extent that the N-terminus has a tyrosyl amino acid. These compounds will for the most part have the following formula:
  • Tyr is tyrosine
  • AA 1 , AA 2 and AA 5 are hydrophobic amino acids having hydrogen or a hydrocarbon side chain of from 1 to 8, usually 1 t 4 carbon atoms, and may be either D or L configuration, preferably only one is D configuration, particularly AA 1 ; preferably, AA 1 , AA 2 and AA 5 have hydrogen (glycine) or an alkyl group of from 1 to 2 carbon atoms, preferably 1 carbon atom (alanine);
  • AA 3 is a hydrophobic amino acid, normally having a hydrocarbon substituent of from 2 to 8, usually 6 to 8 carbon atoms, particularly phenyl;
  • AA 4 is a hydrophobic amino acid of from 3 to 6 carbon atoms normally having a side chain having from 0 to 1 chalcogen heteroatom of atomic number 8 to 16, normally bonded solely to carbon, and is generally alkyl of from 1 to 4 carbon atoms, particularly 4 carbon atoms, alkylthioalkylene or alkoxyalkylene, wherein the alkyl substituent is usually of from 1 to 2 carbon atoms, more usually 1 carbon atom, and the alkylene group is of 1 to 3 carbon atoms, usually 1 to 2 carbon atoms e.g. leucine and methionine;
  • r and r 1 are 0 to 1, usually 1, while r 2 is 0 to 1, usually 0;
  • W a is H for agonist activity and alkyl or cyclopropylmethyl for antagonist activity
  • W 2 is hydroxyl, amino or an amino acid or polypeptide of not more than about 10, usually 4, amino acids which does not adversely affect the opioid activity of the subject oligopeptides.
  • oligopeptides have the following formula: ##STR4## wherein: AA 5-10 are hydrophobic amino acids being unsubstituted at the ⁇ position or having one substituent which is hydrocarbon or having a single chalcogen atom (O or S) and of from 1 to 8, usually 1 to 6, more usually 1 to 4 carbon atoms, when aliphatic and 6 to 8 carbon atoms when aromatic; being normally aliphatic, usually free of aliphatic unsaturation, either straight chain or branched, the chalcogen atom bonded solely to carbon and hydrogen, usually carbon, particularly AA 7 may be alkoxy or alkylthioalkylene of from 2 to 6, usually 3 to 4 carbon atoms;
  • the preferred amino acids which come within the above definition include glycine, alanine, valine, leucine, isoleucine and methionine. Less preferred amino acids include phenylalanine, serine, and threonine. That is, those amino acids which are substantially neutral; one or more of AA 5-10 may be of the D-configuration, for example AA 5-6 ;
  • D g to D k are alkylene of from 2 to 6, usually 2 to 4 carbon atoms, more usually 3 to 4 carbon atoms.
  • Y g to Y k are amino or guanidinyl, either as the amine or its salt;
  • W a and W 2 have been defined previously;
  • r 3-5 are 0 or 1, preferably r 3 being 0, and r 4 being 1, and r 5 being 0.
  • one or more of the basic amino acids may be substituted by histidine, tryptophan or other amino acid, natural or unnatural, which would provide some or equivalent basicity to the naturally occurring basic amino acids, lysine and arginine.
  • one or more other amino acids may be substituted for the hydrophobic amino acids, such as cysteine, threonine, or serine.
  • the hydrocarbon group is preferred.
  • one or more peptide bonds may be modified, particularly the peptide bond of a basic amino acid to a non-basic amino acid, where the basic amino acid follows a basic amino acid in going from the N-terminus. Modification could also include reducing the peptide link to a methyleneamine or similar link.
  • AA 5 is glycine, D- or L-analine
  • AA 6 is glycine
  • AA 7 is leucine or methionine
  • r 3 and r 5 are 0;
  • r 4 is 1;
  • D g , D h , and D i are trimethylene and Y g , Y h , and Y i are guanidinyl;
  • D j and D k are tetramethylene and Y j and Y k are amino;
  • AA 10 is leucine
  • W a is hydrogen
  • W 2 is hydroxyl or amino.
  • compositions because of their high basicity may be combined with polypeptides which have reciprocal amino acids, that is, aspartic or glutamic acid.
  • the acidic amino acids would be linked to hydrophobic amino acids and the resulting oligopeptide acid groups appropriately spaced with hydrophobic amino acids to associate with the basic groups of the subject oligopeptides.
  • Such compositions could be used in combination with the subject oligopeptides, for example, a sextet combined with an enkephalin and the oligopeptide designates as W.
  • the acidic oligopeptide could be linked by a peptide link at the C-terminus, particularly through 1 to 2 basic amino groups.
  • AA 11-13 are glycine or aliphatically substituted amino acids, having alkyl groups from 1 to 6, usually one 1 three 3 atoms at the ⁇ position;
  • R j-n are alkylene of from 1 to 2 carbon atoms, desirably the number of carbon atoms of the complementary D and R groups being a total of 6, that is, lysine would be complementary to aspartic acid, while arginine would be complementary to glutamic acid; and
  • r 6-8 are 0 or 1.
  • compositions may be readily prepared by conventional techniques, for example, a solid state technique performed on a resin support. See Steward and Young, Solid-Phase Peptide Synthesis, W. H. Freeman Co., San Francisco (1969) and Merrifield, J. Am. Chem. Soc. 85 2149-2154 (1963). Conveniently, the oligopeptides may be synthesized automatically employing a Beckman Model 990 Peptide Synthesizer, available from the Spinco Division of Beckman Instruments, Inc., described in Instruction Manual No. SY-IM-2, December 1972.
  • the compounds of the subject invention may be used for preparing antisera for use in immunoassays employing labeled oligopeptides.
  • the oligopeptides may be conjugated to an antigen by means of dialdehydes, particularly of from 4 to 6 carbon atoms and aliphatic.
  • the oligopeptides may be labeled with a variety of labels which are conventionally employed in the literature. Illustrative labels which may be found in patents, are radioactive tags, such as 125 I or 3 H, enzymes, fluorescers or the like. See for example U.S. Pat. Nos. 3,766,162, 3,817,837 and 3,998,943, the appropriate portions of which, describing the labeling and immunoassay, are incorporated herein by reference.
  • the compound is referred to as dynorphin.
  • the desalted material was subjected to reverse phase chromatography on HPLC (C 18 column) using a 10-50% acetonitrile gradient in 5 mM TFA.
  • the peak of biologic activity had no measurable absorbance at 280 nm.
  • Microsequencing of the peptide material was performed by the spinning cup procedure. Hunkapiller & Hood (1978) Biochemistry 17, 2124. An unambiguous sequence was obtained with one-third of the total material, for 13 residues. The amount of peptide, estimated from the first few cycles, was about 400 pmoles, and thus, the potency in the bioassay was approximately 1/3 of 334/0.400, or 267 times that of normorphine. Presence of a blocked contaminant was confirmed in the composition data, which gave distinctly non-integer ratios.
  • the tridecapeptide was synthesized with a sequence corresponding to porcine dynorphin-(1-13): H-tyr-gly-gly-phe-leu-arg-arg-ile-arg-pro-lys-leu-lys-OH.
  • register-shift peptides Two related peptides (referred to here as "register-shift peptides") were also synthesized. These were the des-arg 6 dodecapeptide from dynorphin-(1-13), and the gly 6 tetradecapeptide derivative, in which gly is inserted between leu 5 and arg 6 . Standards of purity were the same as noted above for the tridecapeptide.
  • the guinea pig ileum myenteric plexus-longitudinal muscle preparation was used as described in Kosterlitz et al, supra, and Schulz & Goldstein, supra.
  • a determination of IC50 was done by bracketing, with at least one concentration giving more than, and one giving less than 50% inhibition of the electrically stimulated twitch, and interpolating on a log concentration-percent inhibition plot.
  • Geometric means of IC50 values from several muscle strips were obtained, with standard errors in log units.
  • K d values for naloxone as an antagonist were determined from the ratio of IC50 values in the presence and absence of a fixed naloxone concentration.
  • the mouse vas deferens was used as described in Hughes at al (1975) Brit. J. Pharmacol. 53, 371.
  • IC50 and naloxone K d values were determined as above. In both bioassays, volumes of methanol-HCl solvent up to 20 ⁇ l in the 5-ml tissue bath were without
  • the radioreceptor binding assay used well washed rat brain membranes that had been incubated for 30 min at 37° with 100 mM NaCl to remove bound endogenous ligands. Blume (1978) Proc. Natl. Acad. Sci. 75, 1713. Several radioligands (1 nM final concentration) and competing ligands were tested. Competing ligands were added to the membrane suspension in Tris buffer, pH 7.4 at 23°, and radioligand was added immediately thereafter. Assay volume was 500 ⁇ l. After incubation (1 h, 23°), the mixture was chilled, filtered, washed twice, and counted.
  • 125 -I-dynorphin-(1-13) was prepared by a modification of the method of Hunter and Greenwood, (1962), Nature 194, 495, and separated from unreacted iodide on a Sephadex G-15 column with 0.1% bovine serum albumin in 0.25 M acetic acid. Peptide degradation in vivo and by rat brain membranes was estimated by a shift in the radioactivity peak on a Bio-Gel P-2 column (1.2 ⁇ 41 cm) using n-butanol:acetic acid:water (2:1:4).
  • radioimmunoassay two different antisera were used, raised to leucine-enkephalin in rabbits. See Watson et al (1979) Endorphins in Mental Health Research eds. Usdin, Bunney & Kline (Oxford University Press, New York). Antisera were used at final dilutions required to bind about 30% of 125 -I-leucine-enkephalin in the absence of a competing ligand. This radioligand was prepared by the chloramine-T method (Hunter and Greenwood (1962) Nature 194, 495) and purified on DEAE-Sephadex.
  • IC50 values were obtained from assays spanning the 50% inhibition response, plotted on log dose-percent inhibition plots.
  • IC50 values are geometric means, with standard errors in log units, number of tissue preparations in parentheses. K d determinations were based on four tissue preparations.
  • RX-1 is des-arg 6 -dynorphin-(1-13);
  • RS-2 has gly 6 insertion in dynorphin-(1-13).
  • the natural and synthetic dynorphin is found to be substantially more active than the enkephalins and ⁇ -endophin in the guinea pig ileum test.
  • the dynorphin is found to be about 1000x greater potency than leu-enkephalin, about 350x greater potency than normorphine and about 30x more potent than ⁇ -endorphin.
  • the compound is relatively resistant to treatment with cyanogen bromide and has long persistence in the guinea pig ileum test, unlike ⁇ -endorphin.
  • the molecular weight is found to be about 1750.
  • compositions of this invention can be administered to a mammalian host e.g. domestic animals, and man, in the same manner as other opioid agonists e.g. morphine, are administered.
  • the administered dosage will range from about 0.05 to 40 mg, more usually about 0.5 to 20 mg per 70 kg body weight.
  • the drug may be administered neat, admixed with physiologically acceptable powders or dissolved in physiologically acceptable liquid e.g. water or aqueous ethanol, usually having not more than about 60 volume percent ethanol.
  • physiologically acceptable liquid e.g. water or aqueous ethanol, usually having not more than about 60 volume percent ethanol.
  • the drug may be administered orally, by inhalation, or parenterally e.g. subcutaneously, intravenously or intramuscularly. Concentration of the drug in admixtures or solutions will generally range from about 0.5 to 50 weight percent, usually from about 1 to 30 weight percent.
  • the subject drugs may be used individually or in combination with other drugs, normally employed in conjunction with analgesics.
  • These drugs include aspirin and L-dopa.
  • the subject drugs When administered as a solid, the subject drugs may be administered as pills, capsules, powders, or the like.
  • the subject dynorphin was used for the development of immunoassays, illustrated particularly by a radioimmunoassay.
  • immunoassay it is necessary to prepare antigens which specifically recognize the oligopeptide, in this case dynorphin.
  • dynorphin thyroglobulin was conjugated to synthetic dynorphin (1-13). The procedure for preparing the antigen conjugate, injecting the antigen conjugate into animals and isolating the antisera is described as follows.
  • Synthetic dynorphin (1-13) (3.25 ⁇ moles, 5.2 mg) was dissolved in 2 ml of 100 mM sodium phosphate buffer at neutrality. To the mixture was added with agitation 10 5 cpm of 125 I-dynorphin to serve as a tracer. After addition of the tracer, 26 mg of thyroglobulin (bovine type 1, Sigma) was added and the mixture cooled in an ice-bath after the thyroglobulin dissolved.
  • thyroglobulin bovine type 1, Sigma
  • reaction mixture was dialyzed against 0.1 M sodium phosphate buffer, pH7.4, containing 0.45% NaCl for 5 hrs. in the cold, followed by dialysis overnight against 0.9% NaCl and then against distilled water (3 ⁇ 5 hrs.).
  • the tracer dynorphin (1-13) having 125 I was prepared as follows.
  • the following reagents were prepared: sodium 125 I (17 Ci/mgI) in 5 ⁇ l batches in 0.1 N sodium hydroxide was diluted with 0.1 M sodium phosphate buffer, pH7.4, to make 1 mCi/25 ⁇ l solutions, including the HCl added to neutralize the sodium hydroxide.
  • Chloramine-T (Sigma) was made 0.5 mg/ml in 0.1 M sodium phosphate buffer, pH7.4.
  • Buffer A 0.150 M sodium phosphate, pH7.4;
  • Buffer B buffer A containing 0.1% bovine serum albumin and 0.1% Triton X-100;
  • Antiserum A 1:10 dilution of the antiserum in 50 mM sodium phosphate buffer, pH7.4, containing 50 mM NaCl was diluted 1:10 4 with buffer B;
  • Dynorphin (1-13) standard Standards were prepared by taking 10 ⁇ l of a 10 mM stock solution in water and adding 990 ⁇ l of methanolic HCl to make a 100 mM dynorphin solution followed by 1:100 dilutions to provide the desired series.
  • 125 I-dynorphin (1-13) trace The trace stored in 0.25 M acetic acid containing 0.1% BSA was diluted to about 5,000 cpm/100 ⁇ l with buffer B.
  • the procedure for the assay was as follows: All solutions were in ice at the outset and all operations were carried out in ice. In a 13 ⁇ 100 mM borosilicate glass tube was added in the following order 100 ⁇ l of dynorphin (1-13) standard or test sample in methanolic HCl; 100 ⁇ l of the antiserum dilution; and 100 ⁇ l of the trace, followed by incubation at 0°-4° for 24 hrs. Incubation was terminated with a charcoal mixture prepared by mixing 3 g Norit A, 0.3 g dextran, 15 ml horse serum and the mixture brought to 100 ml with buffer A and mixed for 10 min in a cold room before use.
  • the antiserum recognizes the area of the dynorphin (1-13) at about the 5-6 amino acids from the N-terminus.
  • the subject antiserum is specific for dynorphin (1-13) and is able to distinguish leu-enkephalin.
  • compositions can be used to prepare a sensitive assay for a natural analgesic, by the preparation of a specific, strongly binding antiserum and a labeled analog of the dynorphin.
  • a radioactive label other labels may be employed which include fluorescers, enzymes, or other convenient label.
  • the subject opioid activity containing compounds also find use as standards in testing for binding to opioid receptor sites for other drugs and investigating the secondary and tertiary structural characteristics of opioid receptor binding sites.
  • the subject products find use as a pharmaceutical product, in both research and commercial applications.
  • subject compounds can be used as precursors to potentiate the binding properties of known analgesic compounds.
  • the subject invention greatly extends the ability to achieve analgesic effects by employing natural products or modified natural products. In this way, pain can be alleviated with minimal interference with natural processes, without introduction of undersirable side products and without causing side effects.
  • the subject compositions offer directions to new drugs or other means to provide opiate antagonists or agonists. By potentiating the opioid compounds, lower concentrations will be required, so as to potentially reduce side effects.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pain & Pain Management (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Toxicology (AREA)
  • Emergency Medicine (AREA)
  • Endocrinology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Neurology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Neurosurgery (AREA)
US06/091,615 1979-11-05 1979-11-05 Novel polypeptide analgesics Expired - Lifetime US4396606A (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
US06/091,615 US4396606A (en) 1979-11-05 1979-11-05 Novel polypeptide analgesics
DE8080303698T DE3066200D1 (en) 1979-11-05 1980-10-20 Polypeptides, antisera containing them and methods of their use
EP80303698A EP0029300B1 (en) 1979-11-05 1980-10-20 Polypeptides, antisera containing them and methods of their use
CA000363326A CA1177825A (en) 1979-11-05 1980-10-27 Polypeptide analgesics
JP15394380A JPS5681550A (en) 1979-11-05 1980-11-04 Novel polypeptide analgesic

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US06/091,615 US4396606A (en) 1979-11-05 1979-11-05 Novel polypeptide analgesics

Publications (1)

Publication Number Publication Date
US4396606A true US4396606A (en) 1983-08-02

Family

ID=22228739

Family Applications (1)

Application Number Title Priority Date Filing Date
US06/091,615 Expired - Lifetime US4396606A (en) 1979-11-05 1979-11-05 Novel polypeptide analgesics

Country Status (5)

Country Link
US (1) US4396606A (enrdf_load_stackoverflow)
EP (1) EP0029300B1 (enrdf_load_stackoverflow)
JP (1) JPS5681550A (enrdf_load_stackoverflow)
CA (1) CA1177825A (enrdf_load_stackoverflow)
DE (1) DE3066200D1 (enrdf_load_stackoverflow)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4462941A (en) * 1982-06-10 1984-07-31 The Regents Of The University Of California Dynorphin amide analogs
US4551273A (en) * 1983-02-01 1985-11-05 Eisai Co., Ltd. Analgesic peptide and process for the preparation thereof
WO1991000736A1 (en) * 1989-07-06 1991-01-24 Arizona Technology Development Corporation Dynorphin analogs specific for kappa opioid receptors
WO1994026296A1 (en) * 1993-05-19 1994-11-24 Torrey Pines Institute For Molecular Studies Novel opioid peptide inhibitors
US5482930A (en) * 1993-06-09 1996-01-09 The Regents Of The University Of California Anti-inflammatory composition and method with des-Tyr dynorphin and analogues
WO1996006626A1 (en) * 1994-08-26 1996-03-07 Lee Nancy M Analgesic method with dynorphin analogues truncated at the n-terminus
US5807827A (en) * 1992-06-12 1998-09-15 Des-Tyr Dynorphin Partnership Des-Tyr dynorphin analogues
US20020132777A1 (en) * 2001-01-17 2002-09-19 Zimmer Robert H. Compositions and methods for enhanced pharmacological activity through oral and parenteral administration of compositions comprising polypeptide drug substances and other poorly absorbed active ingredients
US20030060413A1 (en) * 2001-09-06 2003-03-27 Zimmer Robert H. Derivatives of pseudo-peptides, their preparation and their biological uses
US20040102381A1 (en) * 1998-08-14 2004-05-27 Ekwuribe Nnochiri N. Methods of altering the binding affinity of a peptide to its receptor
US20040186058A1 (en) * 1999-08-06 2004-09-23 Zimmer Robert H. Compositions and methods for enhanced pharmacological activity of compositions comprising peptide drug substances
CN101979093B (zh) * 2001-01-17 2013-04-24 罗伯特·H·齐默尔 含有肽原料药和其它不易吸收的活性成分的组合物及其应用
WO2014190313A2 (en) 2013-05-24 2014-11-27 The Arizona Board Of Regents On Behalf Of The University Of Arizona Dynorphin a analogs with bradykinin receptors specificity for modulation of neuropathic pain

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4481191A (en) * 1983-03-30 1984-11-06 The Regents Of The University Of California Method for controlling blood pressure
EP0147194B1 (en) * 1983-12-22 1989-07-19 Yoshio Hosobuchi Compositions for treating cerebral ischemia

Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4097471A (en) * 1977-05-26 1978-06-27 American Home Products Corporation Polypeptides with morphine-like activity
US4098778A (en) * 1977-03-11 1978-07-04 Hoffmann-La Roche Inc. β-Endorphin analog
US4103005A (en) * 1977-10-20 1978-07-25 Hoffmann-La Roche Inc. Novel-enkephalin analogs
US4116950A (en) * 1978-01-16 1978-09-26 Hoffmann-La Roche Inc. Analogs of human β-endorphin
US4123523A (en) * 1976-06-21 1978-10-31 Imperial Chemical Industries Limited Analgesic and sedative polypeptides
USRE29842E (en) 1976-03-17 1978-11-21 Hoffmann-La Roche Inc. Untriakontapeptide with opiate activity
US4127527A (en) * 1977-06-16 1978-11-28 Coy David Howard Novel pentadecapeptides, intermediates therefor, and compositions and methods employing said pentadecapeptides
US4127525A (en) * 1977-06-16 1978-11-28 Coy David Howard Novel tridecapeptides, intermediates therefor, and compositions and methods employing said tridecapeptides
US4127526A (en) * 1977-06-16 1978-11-28 Coy David Howard Novel tetradecapeptides, intermediates therefor, and compositions and methods employing said tetradecapeptides
US4127540A (en) * 1977-06-16 1978-11-28 Coy David Howard Novel derivatives of δ-endorphins, intermediates therefor, and compositions and methods employing said derivatives
US4127528A (en) * 1977-06-16 1978-11-28 Coy David Howard Novel derivatives of alpha-endorphin, intermediates therefor and compositions and methods employing said derivatives
US4127538A (en) * 1977-06-16 1978-11-28 Coy David Howard Novel pentacosapeptides, intermediates therefor and pharmaceutical compositions and methods employing said pentacosapeptides
US4143032A (en) * 1977-06-20 1979-03-06 American Home Products Corporation Hexapeptides having analgesic activities
US4148788A (en) * 1978-01-23 1979-04-10 Hoffmann-La Roche Inc. Synthesis of thymosin α1
US4161522A (en) * 1975-04-04 1979-07-17 The Regents Of The University Of California Method for blocking allergic responses
US4178284A (en) * 1978-12-11 1979-12-11 American Home Products Corporation Octapeptides
US4180501A (en) * 1978-06-16 1979-12-25 Coy David Howard Bis (polypeptide) derivatives of enkephalin
US4207311A (en) * 1977-07-18 1980-06-10 The Salk Institute For Biological Studies Peptides having analgesic and thermoregulative properties
US4223017A (en) * 1975-04-15 1980-09-16 Burroughs Wellcome Co. Biologically active amides
US4229438A (en) * 1978-05-12 1980-10-21 Takeda Chemical Industries, Ltd. Nonapeptides
US4230696A (en) * 1972-12-14 1980-10-28 St. Luke's Hospital Polypeptides useful for the purpose of treating multiple sclerosis
US4232008A (en) * 1978-11-17 1980-11-04 Ortho Pharmaceutical Corporation Tetrapeptides and methods
US4250087A (en) * 1979-12-10 1981-02-10 Hoffmann-La Roche Inc. Carboxyl terminus analogs of β-endorphin
US4254107A (en) * 1979-11-27 1981-03-03 Merck & Co., Inc. Long-lasting agonists of enkephalin
US4254106A (en) * 1976-01-26 1981-03-03 Burroughs Wellcome Co. Biologically active amides
US4256736A (en) * 1978-03-02 1981-03-17 Akzona Incorporated Psychopharmacological peptides

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5291839A (en) * 1976-01-26 1977-08-02 Wellcome Found Peptide and derivatives
SE436645C (sv) * 1976-04-29 1996-07-22 Bonnierfoeretagen Ab Antigeniskt aktiv polypeptid, som kan användas vid cancerdiagnosticering och vid framställning av antikroppar

Patent Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4230696A (en) * 1972-12-14 1980-10-28 St. Luke's Hospital Polypeptides useful for the purpose of treating multiple sclerosis
US4161522A (en) * 1975-04-04 1979-07-17 The Regents Of The University Of California Method for blocking allergic responses
US4223017A (en) * 1975-04-15 1980-09-16 Burroughs Wellcome Co. Biologically active amides
US4254106A (en) * 1976-01-26 1981-03-03 Burroughs Wellcome Co. Biologically active amides
USRE29842E (en) 1976-03-17 1978-11-21 Hoffmann-La Roche Inc. Untriakontapeptide with opiate activity
US4123523A (en) * 1976-06-21 1978-10-31 Imperial Chemical Industries Limited Analgesic and sedative polypeptides
US4098778A (en) * 1977-03-11 1978-07-04 Hoffmann-La Roche Inc. β-Endorphin analog
US4097471A (en) * 1977-05-26 1978-06-27 American Home Products Corporation Polypeptides with morphine-like activity
US4127527A (en) * 1977-06-16 1978-11-28 Coy David Howard Novel pentadecapeptides, intermediates therefor, and compositions and methods employing said pentadecapeptides
US4127525A (en) * 1977-06-16 1978-11-28 Coy David Howard Novel tridecapeptides, intermediates therefor, and compositions and methods employing said tridecapeptides
US4127528A (en) * 1977-06-16 1978-11-28 Coy David Howard Novel derivatives of alpha-endorphin, intermediates therefor and compositions and methods employing said derivatives
US4127538A (en) * 1977-06-16 1978-11-28 Coy David Howard Novel pentacosapeptides, intermediates therefor and pharmaceutical compositions and methods employing said pentacosapeptides
US4127540A (en) * 1977-06-16 1978-11-28 Coy David Howard Novel derivatives of δ-endorphins, intermediates therefor, and compositions and methods employing said derivatives
US4127526A (en) * 1977-06-16 1978-11-28 Coy David Howard Novel tetradecapeptides, intermediates therefor, and compositions and methods employing said tetradecapeptides
US4143032A (en) * 1977-06-20 1979-03-06 American Home Products Corporation Hexapeptides having analgesic activities
US4207311A (en) * 1977-07-18 1980-06-10 The Salk Institute For Biological Studies Peptides having analgesic and thermoregulative properties
US4103005A (en) * 1977-10-20 1978-07-25 Hoffmann-La Roche Inc. Novel-enkephalin analogs
US4116950A (en) * 1978-01-16 1978-09-26 Hoffmann-La Roche Inc. Analogs of human β-endorphin
US4148788A (en) * 1978-01-23 1979-04-10 Hoffmann-La Roche Inc. Synthesis of thymosin α1
US4256736A (en) * 1978-03-02 1981-03-17 Akzona Incorporated Psychopharmacological peptides
US4229438A (en) * 1978-05-12 1980-10-21 Takeda Chemical Industries, Ltd. Nonapeptides
US4180501A (en) * 1978-06-16 1979-12-25 Coy David Howard Bis (polypeptide) derivatives of enkephalin
US4232008A (en) * 1978-11-17 1980-11-04 Ortho Pharmaceutical Corporation Tetrapeptides and methods
US4178284A (en) * 1978-12-11 1979-12-11 American Home Products Corporation Octapeptides
US4254107A (en) * 1979-11-27 1981-03-03 Merck & Co., Inc. Long-lasting agonists of enkephalin
US4250087A (en) * 1979-12-10 1981-02-10 Hoffmann-La Roche Inc. Carboxyl terminus analogs of β-endorphin

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4462941A (en) * 1982-06-10 1984-07-31 The Regents Of The University Of California Dynorphin amide analogs
US4551273A (en) * 1983-02-01 1985-11-05 Eisai Co., Ltd. Analgesic peptide and process for the preparation thereof
EP0115850A3 (en) * 1983-02-01 1987-02-04 Eisai Co., Ltd. Novel peptide, process for the preparation thereof and pharmaceutical composition containing said peptide
WO1991000736A1 (en) * 1989-07-06 1991-01-24 Arizona Technology Development Corporation Dynorphin analogs specific for kappa opioid receptors
US5807827A (en) * 1992-06-12 1998-09-15 Des-Tyr Dynorphin Partnership Des-Tyr dynorphin analogues
WO1994026296A1 (en) * 1993-05-19 1994-11-24 Torrey Pines Institute For Molecular Studies Novel opioid peptide inhibitors
US5482930A (en) * 1993-06-09 1996-01-09 The Regents Of The University Of California Anti-inflammatory composition and method with des-Tyr dynorphin and analogues
WO1996006626A1 (en) * 1994-08-26 1996-03-07 Lee Nancy M Analgesic method with dynorphin analogues truncated at the n-terminus
US20040102381A1 (en) * 1998-08-14 2004-05-27 Ekwuribe Nnochiri N. Methods of altering the binding affinity of a peptide to its receptor
US7671029B2 (en) * 1999-08-06 2010-03-02 Immupharma Sa Compositions and methods for enhanced pharmacological activity of compositions comprising peptide drug substances
US8080519B2 (en) 1999-08-06 2011-12-20 Immupharma Sa (France) Compositions and methods for enhanced pharmacological activity of compositions comprising peptide drug substances
US20040186058A1 (en) * 1999-08-06 2004-09-23 Zimmer Robert H. Compositions and methods for enhanced pharmacological activity of compositions comprising peptide drug substances
US20100292158A1 (en) * 1999-08-06 2010-11-18 Immupharma Sa (France) Compositions and Methods for Enhanced Pharmacological Activity of Compositions Comprising Peptide Drug Substances
US20020132777A1 (en) * 2001-01-17 2002-09-19 Zimmer Robert H. Compositions and methods for enhanced pharmacological activity through oral and parenteral administration of compositions comprising polypeptide drug substances and other poorly absorbed active ingredients
US6908900B2 (en) 2001-01-17 2005-06-21 Zimmer & Associates Ag Compositions and methods for enhanced pharmacological activity through oral and parenteral administration of compositions comprising polypeptide drug substances and other poorly absorbed active ingredients
CN101979093B (zh) * 2001-01-17 2013-04-24 罗伯特·H·齐默尔 含有肽原料药和其它不易吸收的活性成分的组合物及其应用
US20030060413A1 (en) * 2001-09-06 2003-03-27 Zimmer Robert H. Derivatives of pseudo-peptides, their preparation and their biological uses
WO2014190313A2 (en) 2013-05-24 2014-11-27 The Arizona Board Of Regents On Behalf Of The University Of Arizona Dynorphin a analogs with bradykinin receptors specificity for modulation of neuropathic pain
US10428115B2 (en) 2013-05-24 2019-10-01 Arizona Board Of Regents On Behalf Of The University Of Arizona Dynorphin A analogs with bradykinin receptors specificity for modulation of neuropathic pain

Also Published As

Publication number Publication date
JPS5681550A (en) 1981-07-03
DE3066200D1 (en) 1984-02-23
EP0029300B1 (en) 1984-01-18
EP0029300A1 (en) 1981-05-27
JPH0224838B2 (enrdf_load_stackoverflow) 1990-05-30
CA1177825A (en) 1984-11-13

Similar Documents

Publication Publication Date Title
US4396606A (en) Novel polypeptide analgesics
Goldstein et al. Dynorphin-(1-13), an extraordinarily potent opioid peptide.
Bissette et al. Neurotensin, a biologically active peptide
CA1241645A (en) Dynorphin amide analogs
Amiche et al. Dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2): a potent and fully specific agonist for the delta opioid receptor.
Shimohigashi et al. Dehydro-enkephalins. IV. Discriminative recognition of delta and mu opiate receptors by enkephalin analogs
CA1094549A (en) Therapeutic insulin preparation and a process for the production of a stable insulin preparation with protracted effect
Goodfriend et al. Immunochemical studies of angiotensin
US5854216A (en) Marker for growth hormone-releasing factor receptors
EP0055113B1 (en) Deca- undeca- dodeca- and tridecapeptides with thymic activity, method for their preparation and compositions containing them
LV10108B (en) Inhibitors of cells proliferation, pharmaceutical composition, process of producing of compounds, methods of inhibition of cellular proliferation
US5017689A (en) Dynorphin analogs specific for kappa opioid receptors
Cockle et al. Specific processing of the thyrotropin‐releasing prohormone in rat brain and spinal cord
US4468383A (en) Dimeric enkephalins
KR20220110789A (ko) 칼슘 감지 수용체 효능제 화합물 및 이의 응용
US5326751A (en) Enkephalin analogs
Nussberger et al. Selectivity of angiotensin II antisera
JP2004002331A (ja) 肝臓癌の治療
Biondi et al. Opioid peptides: synthesis and biological activity of new endomorphin analogues
US5061691A (en) Enkephalin analogs
JPS62286997A (ja) 薬理的に活性のあるペプチッド
Medzihradszky The chemistry of the opioid receptor binding sites: Josef Rudinger Memorial Lecture 2002
US4490363A (en) Metorphamide--a novel morphine-like peptide
FERRARA et al. β‐Endorphin: Interaction of synthetic analogs having different chain lengths with morphine and enkephalin receptors in rat brain membranes
CA2089247A1 (en) Small peptide and pseudopeptide amides inhibiting the proliferation of small-cell and epithelial-cell lung cancer cells

Legal Events

Date Code Title Description
STCF Information on status: patent grant

Free format text: PATENTED CASE

AS Assignment

Owner name: RESEARCH CORPORATION TECHNOLOGIES, INC., ARIZONA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:RESEARCH CORPORATION;REEL/FRAME:005289/0788

Effective date: 19900427

Owner name: RESEARCH CORPORATION, NEW YORK

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:ADDICTION RESEARCH FOUNDATION;REEL/FRAME:005289/0796

Effective date: 19851125